Thanks to the International HapMap Project which decodes the human genome, and especially makes SNP (Single Nucleotide Polymorphism) maps, information on the human genome is increasing more and more. In addition, research is progressing in a wide scale all over the world to find out the association between the thus acquired genomic information and individual constitutions, to understand genetic level variations between individual constitutions, and to realize “medication customized to the genetic information of each individual (personalized medicine)” which enables the diagnosis/treatment/prevention of diseases and the administration of drugs customized to individual traits. The genetic variations referred to herein mean variations in the nucleotide sequence of the genome between individuals, the most common type of variation of which is single nucleotide polymorphism (SNP). Moreover, in these days, it has been understood that variations in the number of repetitions (number of copies) of a short nucleotide sequence (Copy Number Variation: CNV) are widely spread in the whole genome, and also the associations between such CNV variations and diseases have been pointed out.
Here, in order to understand the genetic level variations between individuals, it is necessary to examine the genotypes of respective individuals. For example, a case is assumed in which it has been known that there are three genotypes of AA, AG, and GG in a certain type of SNP, wherein the symbol A represents an adenine base and the symbol G represents a guanine base. This SNP is an example consisting of cases where adenine or guanine comes to a specific position of a given genome. Accordingly, the test for distinguishing genotypes of this SNP is to determine the genotypes of these three variations. In other words, this discrimination can be achieved by seeing whether A accounts for 0 or 100, whether G accounts for 0 or 100, or whether A and G respectively account for 50 and 50. In this way, the detection of germline mutations such as SNP can be said to be substantially qualitative. Various kinds of relatively easy and convenient methods thereof have been adopted in practice.
On the other hand, when it comes to cancer cells, the onset is considered to be such that a somatic level mutation takes place and abnormal growth is triggered by this mutation. Accordingly, some specific kinds of cancer cells show mutations in specific genes. Thus, it is possible to detect such cancer cells by seeing the indication of this kind of mutation. However, cancer cells are so various that it is not always easy to specify cancer cells only by seeing a single kind of mutation.
Moreover, in recent pharmaceutical treatments, medicines which target specific types of molecules (such as a protein) in vivo have been developed, and some of which that can provide high efficacy with small side effects are being discovered. These are called molecularly targeted agents, and are actively developed mainly in the field of cancer treatment. Quite recently, it has been revealed to be impossible for these molecularly targeted agents to exert the efficacy of such medicines in the case where a mutation takes place in a protein on the downstream of the signal transduction of the targeted molecule. In this case, it is becoming possible to predict the efficacy of the medicine by examining a mutation in a gene which encodes the protein where the mutation takes place. A new field of personalized medicine which is different from the SNP detection is being opened.
Most of above-mentioned mutations specific to cancer cells or mutations showing the resistance to molecularly targeted agents are somatic mutations. In a case of the germline mutation mentioned above, every cell shows a common mutation; whereas, in a somatic mutation, only mutated cells show a mutation and non-mutated cells (usually, normal cells) shows no mutation. Accordingly, the situation is usually such that mutated cells and normal cells are mixedly present in the analyte (the specimen serving as the subject to be tested), and the mutant gene and the normal genes are both present in proportion to the abundance ratio of these cells. That is to say, in a case where normal cells account for the majority of the specimen and mutated cells account for only a part of it, it is necessary to detect the mutant gene which is scantily present within a large population of the normal gene. This differs from the detection of a germline mutation, and makes it more difficult to detect a somatic gene mutation.
The approach for detecting a somatic gene mutation is largely classified into two methods. One is a method to discriminate the normal gene and the mutant gene at a stage of the gene amplification. Concretely, this is a method to specifically amplify only the mutant gene.
For example, the method deemed to be most sensitive is a so-called “mutant-enriched PCR” method in which only the normal gene is cleaved using a restriction enzyme and only the non-cleaved mutant gene is amplified (for example, refer to Non-patent Document 1). This method is said to be capable of detecting the mutant gene at a concentration of one molecule per 106 normal gene molecules through repetition of reactions to amplify the mutant gene (for example, refer to Non-patent Document 2). This method is excellent in terms of high sensitivity in this way. However, it is not a method applicable to general diagnoses because the manipulation is quite complicated.
In addition, a method has been developed in which, in PCR or such a primer extension reaction, the amplification is performed after discriminating a single nucleotide difference. This method is called “ARMS (amplification refractory mutation system)” (for example, refer to Non-patent Document 3), “ASPCR (allele specific PCR)” (for example, refer to Non-patent Document 4), or the like. This method is excellent as it is relatively highly sensitive, requires no manipulation other than usual PCR amplification reactions, is capable of carrying out all the reactions in a closed system, is very easy and convenient, and is free from PCR carry-over contamination. However, this can also be said to be a method involving a high risk of quasi-positive, because, even once a normal gene has been accidentally amplified by misdistinguishing a single nucleotide, the normal gene would be amplified in the following amplification reactions afterwards likewise of the mutant gene amplification. When adopting this method, it is necessary to strictly control the reaction conditions, namely, the reaction temperature, the salt concentration, and the like, and it is also necessary to strictly control the amount of the template (for example, refer to Non-patent Document 5). So, this method is not suitable for clinical tests where a large number of unspecified analytes have to be examined, nor diagnosis methods that should be easy and convenient as well as being highly accurate.
An other approach for detecting a somatic gene mutation is a method in which the mutant gene and the normal gene are concurrently amplified, and thereafter the mutant gene and the normal gene are discriminated and detected. The method for discriminating and detecting the thus amplified mutant gene and normal gene can be exemplified by various kinds of methods using electrophoresis, methods using hybridization, and the like (for example, refer to Non-patent Document 5). However, in most of these methods, it is difficult to accurately detect a small population of mutant gene contained in a large population of normal gene. For example, the method deemed to be the gold standard for detecting a mutant gene is a dideoxy sequencing method. The dideoxy sequencing method is capable of detecting a mutant gene with a relatively high sensitivity. Nonetheless, in cases where the mutant gene and the normal gene are mixedly present, the detection sensitivity for the mutant gene is about 10%, meaning that detection with a very high sensitivity is not feasible. Besides, it is reported that a pyrosequencing method is capable of increasing the detection sensitivity up to about 5%, and is superior to the dideoxy sequencing method (for example, refer to Non-patent Document 6).
Also developed is a method in which a mutation-including sequence is amplified by PCR, then the melting curve of the double-stranded DNA of the amplicon is obtained, and the ratio of the mutant gene is calculated from the difference in the melting curve between the mutant gene and the normal gene. This method is also considered to be capable of detecting a mutant gene contained in a mass of the normal gene with a sensitivity up to about 5% (for example, refer to Non-patent Document 7).
In addition, a PCR-PHFA method which utilizes a strand exchange reaction between two strands having homologous nucleotide sequences (strand displacement reaction) has been developed. The PCR-PHFA method is a method to detect a mutation by utilizing the following phenomenon such that: if the nucleotide sequences of a sample whose genotype is to be distinguished (double-stranded nucleic acid) and a reference double-stranded nucleic acid whose sequence is already known are completely identical, respective strands can not be discriminated and a strand exchange (strand displacement) takes place therebetween; whereas, if these are not identical even only by a single nucleotide difference, strands having completely homologous nucleotide sequences are preferentially paired to form a duplex, and therefore the exchange would not take place between the sample and the reference double-stranded nucleic acid. By using this PCR-PHFA method, it is reportedly possible to detect a mutant gene in the actual analyte with a high sensitivity of about 1% (for example, refer to Non-patent Document 8). In this way, the PCR-PHFA method is a highly reproducible method with a high detection sensitivity. However, the manipulation is a little complicated (for example, refer to Patent Document 1) and also involves carry-over contamination and such problems. In order to solve these problems, several types of improved methods have been proposed.
For example, Patent Document 2 discloses a method as an improved PCR-PHFA method which utilizes fluorescence resonance energy transfer. In PCR-PHFA methods for accurately measuring a very small population of mutant gene with a high sensitivity, it is necessary to detect a strand exchange between two double-stranded nucleic acids having homologous nucleotide sequences, in many cases of which, however, the sample double-stranded nucleic acid is not labeled, while the reference nucleic acid whose sequence is already known to be subjected to the strand exchange is labeled. In the method of Patent Document 2, a vicinity of the 5′ end of one strand of the reference nucleic acid is labeled by binding a fluorescent substance, and a vicinity of the 3′ end of the other strand is labeled with a different fluorescent substance. If no strand displacement reaction takes place and the reference nucleic acid remains as the initial duplex, a fluorescence resonance energy transfer between two different fluorescent substances is observed. In contrast, if a strand displacement reaction with the sample double-stranded nucleic acid takes place, no fluorescence resonance energy transfer is observed. Accordingly, the level of strand exchange can be assessed by measuring the level of this fluorescence resonance energy transfer.
Meanwhile, recent gene detection technologies are remarkably progressing, and methods for simultaneously detecting expressions or mutations of a large number of genes have been developed as an ensemble of a minute processing technique and a fluorescence detection method. Highly sensitive detection for a mutant gene that can be combined with these technologies has been desired. Furthermore, by conducting PCR-PHFA in a reaction vessel as a closed system rather than in a tube, the risk of contamination can be drastically reduced, and application to easy, convenient, and quick nucleic acid tests is possible.